摘要
目的 克隆MDR1及TNF-α基因cDNA，构建反向pEGFP-MDR1重组表达载体和pDsRed2-TNF-α重组表达载体，并在乳腺癌耐药细胞株MCF-7／ADR中进行表达。方法 应用RT-PCR和DNA重组技术构建反向绿色荧光蛋白pEGFP-MDR1融合蛋白表达载体和红色荧光蛋白pDsRed2-TNF-α融合蛋白表达载体，经脂质体分别和同时导入乳腺癌耐药细胞株MCF-7／ADR中进行表达，应用荧光显微镜直接观察融合蛋白的表达情况，G418筛选阳性克隆。结果 转染了反向pEGFP-MDR1载体的MCF-7／ADR细胞在胞浆和胞核均可见到绿色荧光。而转染了pDsRed2-TNF-α载体的MCF-7／ADR细胞在胞浆和胞核均可见到红色荧光。而同时转染了反向pEGFP-MDR1载体和pDsRed2-TNF-α载体的细胞胞浆和胞核均可见到红色及绿色荧光。结论 反向pEGFP-MDR1融合蛋白和pDsRed2-TNF-α融合蛋白能在乳腺癌耐药细胞MCF-7／ADR中分别及同时获得表达，为进一步研究基因治疗联同免疫治疗逆转乳腺癌耐药建立了很好的基础。
Objective To express the recombinant vectors of reverse pEGFP-MDR1 and pDsRed2-TNF-α in multidrug resistant breast cancer cell line MCF-7/ADR. Methods To Construct the recombinant vector of enhanced green fluorescent protein（ EGFP） with reverse MDR1 gene, as well as recombinant vector of red fluorescent protein（DsRed2） with TNF-α gene by RT-PCR and DNA recombinant techniques.The recombinant vectors were introduced into multidrug resistant breast cancer cells with lipofectin.The expression of the fusion proteins were observed by fluorescent mlcroscope.The positive cell clones were screened by G418 reagent.Results Cells transfected with reverse pEGFP-MDR1 vector can be seen with green fluorescence in cytoplasm and nuclei. Cells transfected with pDsRed2- TNF-α vector can be seen with red fluorescence. Ceils transfected with both reverse pEGFP-MDR1 and pDsRed2-TNF-α vectors can be seen with both green and red fluorescence.Conclusion The expression of recombinant vectors of reverse pEGFP-MDR1 and pDsRed2- TNF-α in multidrug resistant breast cancer cell line MCF-7/ADR provides a good foundation for further research on reversal of multidrug resistance in breast cancer by genetherapy combined with immunotherapy.
出处
《医学文选》
2006年第2期175-177,共3页
Anthology of Medicine
基金
深圳市科技局课题（JH200505260318A）
